Original Enzyme Activity Research Articles

Oral self-emulsifying delivery systems for systemic administration of therapeutic proteins: science fiction?

Objective: The aim of this study was to develop self-emulsifying drug delivery systems (SEDDS) for oral delivery of therapeutic proteins through hydrophobic ion pairing.Method: Horseradish peroxidase (HRP), a model protein, was ion paired with sodium docusate to increase its hydrophobicity. The formed enzyme - surfactant complex was incorporated into SEDDS, followed by permeation studies across Caco-2 cell monolayer and freshly excised rat intestine.Results: Hydrophobic ion pairs (HIP) were formed between HRP and sodium docusate with the efficiency of 87.49 ± 1.35%. The formed complex maintained 60.97 ± 1.48% of the original enzyme activity. The ion pair was subsequently loaded into SEDDS with a payload of 0.1% (mass per cent, m/m). The obtained emulsion formed by SEDDS had a droplet size in the range from 20 to 200 nm with negative zeta potential. Permeation mechanism of the enzyme was energy-dependent and the encapsulation of the HIP complex in SEDDS enhanced the permeation of the enzyme through the Caco-2 cell monolayer and freshly excised rat intestine by 4 times and 2.5 times compared to the free enzyme, respectively.Conclusion: According to these findings, hydrophobic ion pairing followed by incorporation to SEDDS might be considered as a potential strategy for oral delivery of therapeutic proteins.

User-Tailored Metal-Organic Frameworks as Supports for Carbonic Anhydrase.

Carbonic anhydrase (CA) was previously proposed as a green alternative for biomineralization of carbon dioxide (CO2). However, enzyme's fragile nature when in synthetic environment significantly limits such industrial application. Herein, we hypothesized that CA immobilization onto flexible and hydrated "bridges" that ensure proton-transfer at their interfaces leads to improved activity and kinetic behavior and potentially increases enzyme's feasibility for industrial implementation. Our hypothesis was formulated considering that water plays a key role in the CO2 hydration process and acts as both the reactant as well as the rate-limiting step of the CO2 capture and transformation process. To demonstrate our hypothesis, two types of user-synthesized organic metallic frameworks [metal-organic frameworks (MOFs), one hydrophilic and one hydrophobic] were considered as model supports and their surface characteristics (i.e., charge, shape, curvature, size, etc.) and influence on the immobilized enzyme's behavior were evaluated. Morphology, crystallinity and particle size, and surface area of the model supports were determined by scanning electron microscopy, dynamic light scattering, and nitrogen adsorption/desorption measurements, respectively. Enzyme activity, kinetics, and stability at the supports interfaces were determined using spectroscopical analyses. Analysis showed that enzyme functionality is dependent on the support used in the immobilization process, with the enzyme immobilized onto the hydrophilic support retaining 72% activity of the free CA, when compared with that immobilized onto the hydrophobic one that only retained about 28% activity. Both CA-MOF conjugates showed good storage stability relative to the free enzyme in solution, with CA immobilized at the hydrophilic support also revealing increased thermal stability and retention of almost all original enzyme activity even after heating treatment at 70 °C. In contrast, free CA lost almost half of its original activity when subject to the same conditions. This present work suggests that MOFs tunable hydration conditions allow high enzyme activity and stability retention. Such results are expected to impact CO2 storage and transformation strategies based on CA and potentially increase user-integration of enzyme-based green technologies in mitigating global warming.

"Tag and Modify" Protein Conjugation with Dynamic Covalent Chemistry.

The development of small protein tags that exhibit bioorthogonality, bond stability, and reversibility, as well as biocompatibility, holds great promise for applications in cellular environments enabling controlled drug delivery or for the construction of dynamic protein complexes in biological environments. Herein, we report the first application of dynamic covalent chemistry both for purification and for reversible assembly of protein conjugates using interactions of boronic acid with diols and salicylhydroxamates. Incorporation of the boronic acid (BA) tag was performed in a site-selective fashion by applying disulfide rebridging strategy. As an example, a model protein enzyme (lysozyme) was modified with the BA tag and purified using carbohydrate-based column chromatography. Subsequent dynamic covalent "click-like" bioconjugation with a salicylhydroxamate modified fluorescent dye (BODIPY FL) was accomplished while retaining its original enzymatic activity.

Optimization, characterization and thermodynamic studies on B. licheniformis ALW1 keratinase

Optimization of B. licheniformis ALW1 keratinase was investigated by using a Plackett – Burman design (PBD) and Central Composite Design (CCD). PBD showed that galactose, inoculum size and corn steep liquorwere the most effectivevariables played a rolein improving the enzyme productivity (87.65U/mL). CCD results recorded an increase in enzyme productivity to about1.4-fold compared to the basal medium (99.1 U/mL). The optimum activity for the partial purified enzyme was obtained at pH 8.5 and 70˚C. The activation and deactivation energy were calculated to be25.37 kJmol-1 and73.38 kJmol-1 respectively. The half-life time was 1380,690,530, and383 min. at 50˚C,55˚C,60˚C and 65˚C respectively. Also, D values were 4600,2300,1769, 1277min. at the same degree respectively. ∆G° (kJmol-1) kept relatively constant between 50-60˚C (191.49 kJmol-1-193.31 kJmol-1) and noticeably increase at 65˚C (212.86 kJmol-1). ∆H° (kJmol-1) recorded minor decrease by the increase of temperature. Approximately, most of the tested metals ions have stimulation effect in enzyme activityand MgSO4.H2O was the best (146%). Among all the tested detergents tween 80 retained 97% of original enzyme activity. DMSO increased the enzyme activity about 11%, while propanol and acetonitrile reduced the enzyme activity to about 14% and 10% respectively. All the reducing agents had a stimulating effect on enzyme activity with variable degrees. The enzyme (980 U) had the ability to hydrolyze 74% of the feather to nutritional valuable protein.

Effects of Laser Radiation on Mitochondria and Mitochondrial Proteins Subjected to Nitric Oxide.

The biological roles of heme and nonheme nitrosyl complexes in physiological and pathophysiological conditions as metabolic key players are considered in this study. Two main physiological functions of protein nitrosyl complexes are discussed—(1) a depot and potential source of free nitric oxide (NO) and (2) a controller of crucial metabolic processes. The first function is realized through the photolysis of nitrosyl complexes (of hemoglobin, cytochrome c, or mitochondrial iron–sulfur proteins). This reaction produces free NO and subsequent events are due to the NO physiological functions. The second function is implemented by the possibility of NO to bind heme and nonheme proteins and produce corresponding nitrosyl complexes. Enzyme nitrosyl complex formation usually results in the inhibition (or enhancement in the case of guanylate cyclase) of its enzymatic activity. Photolysis of protein nitrosyl complexes, in this case, will restore the original enzymatic activity. Thus, cytochrome c acquires peroxidase activity in the presence of anionic phospholipids, and this phenomenon can be assumed as a key step in the programmed cell death. Addition of NO induces the formation of cytochrome c nitrosyl complexes, inhibits its peroxidase activity, and hinders apoptotic reactions. In this case, photolysis of cytochrome c nitrosyl complexes will reactivate cytochrome c peroxidase activity and speed up apoptosis. Control of mitochondrial respiration by NO by formation or photolytic decay of iron–sulfur protein nitrosyl complexes is an effective instrument to modulate mitochondrial metabolism. These questions are under discussion in this study.

Biochemical characterization and low-resolution SAXS structure of an exo-polygalacturonase from Bacillus licheniformis

Among the structural polymers present in the plant cell wall, pectin is the main component of the middle lamella. This heterogeneous polysaccharide has an α-1,4 galacturonic acid backbone, which can be broken by the enzymatic action of pectinases, such as exo-polygalacturonases, that sequentially cleave pectin from the non-reducing ends, releasing mono or di-galacturonic acid residues. Constant demand for pectinases that better suit industrial requirements has motivated identification and characterization of novel enzymes from diverse sources. Bacillus licheniformis has been used as an important source for bioprospection of several industrial biomolecules, such as surfactants and enzymes, including pectate lyases. Here we cloned, expressed, purified, and biochemically and structurally characterized an exo-polygalacturonase from B. licheniformis (BlExoPG). Its low-resolution molecular envelope was derived from experimental small-angle scattering data (SAXS). Our experimental data revealed that BlExoPG is a monomeric enzyme with optimum pH at 6.5 and optimal temperature of approximately 60°C, at which it has considerable stability over the broad pH range from 5 to 10. After incubation of the enzyme for 30min at pH ranging from 5 to 10, no significant loss of the original enzyme activity was observed. Furthermore, the enzyme maintained residual activity of greater than 80% at 50°C after 15h of incubation. BlExoPG is more active against polygalacturonic acid as compared to methylated pectin, liberating mono galacturonic acid as a unique product. Its enzymatic parameters are Vmax=4.18μM.s−1,Km=3.25mgmL−1 and kcat=2.58s−1.

Co-immobilization of cellulase and laccase onto the reversibly soluble polymers for decolorization of denim fabrics

Both cellulase and laccase can be used in the denim washing to produce the desired aged look in the indigo-dyed denim garments. In this study, cellulase and laccase were co-immobilized onto the reversibly soluble polymers (Eudragit S-100 or Eudragit L-100). Futher, the molecular weight distribution and the enzymatic properties of Eudragit-cellulase-laccase were investigated. The results showed that Eudragit-cellulase-laccase exhibited higher molecular weights than the native cellulase or laccase, and showed greater stability than the native enzymes at higher pH and temperature. Eudragit-cellulaselaccase could retain more than 35 % of the original enzyme activities after five cycles of repeated uses, showing certain reusability. Moreover, the effects of Eudragit-cellulase-laccase treatments on denim washing were also investigated. The decoloration effects of the denim fabrics treated with Eudragit-cellulase-laccase were found to be almost similar to those of the denim fabrics treated with the native cellulase and laccase together. However, the denim fabrics treated with Eudragit-cellulase-laccase showed higher tensile strength than those treated with the native cellulase used alone or the native cellulase and laccase used together.

Correlation of Conformational Changes and Protein Degradation with Loss of Lysozyme Activity Due to Chlorine Dioxide Treatment.

Chlorine dioxide (ClO2) is a potent oxidizing agent used for the treatment of drinking water and decontamination of facilities and equipment. The purpose of this research is to elucidate the manner in which ClO2 destroys proteins by studying the effects of ClO2 on lysozyme. The degree of enzyme activity lost can be correlated to the treatment time and levels of the ClO2 used. Lysozyme activity was drastically reduced to 45.3% of original enzyme activity when exposed to 4.3mM ClO2 in the sample after 3h. Almost all activities were lost in 3h after exposure to higher ClO2 concentrations of up to 16.8 and 21.9mM. Changes in protein conformation and amount as a result of ClO2 treatment were determined using the Raman spectroscopy and gel electrophoresis. Raman shifts and the alteration of spectral features observed in the ClO2-treated lysozyme samples are associated with loss of the α-helix secondary structure, tertiary structure, and disulfide bond. Progressive degradation of the denatured lysozyme by increasing levels of chlorine dioxide was also observed in gel electrophoresis. Hence, ClO2 can effectively cause protein denaturation and degradation resulting in loss of enzyme activity.

Microbial surface displaying formate dehydrogenase and its application in optical detection of formate.

In the present work, NAD(+)-dependent formate dehydrogenase (FDH), encoded by fdh gene from Candida boidinii was successfully displayed on Escherichia coli cell surface using ice nucleation protein (INP) from Pseudomonas borealis DL7 as an anchoring protein. Localization of matlose binding protein (MBP)-INP-FDH fusion protein on the E. coli cell surface was characterized by SDS-PAGE and enzymatic activity assay. FDH activity was monitored through the oxidation of formate catalyzed by cell-surface-displayed FDH with its cofactor NAD(+), and the production of NADH can be detected spectrometrically at 340nm. After induction for 24h in Luria-Bertani medium containing isopropyl-β-d-thiogalactopyranoside, over 80% of MBP-INP-FDH fusion protein present on the surface of E. coli cells. The cell-surface-displayed FDH showed optimal temperature of 50°C and optimal pH of 9.0. Additionally, the cell-surface-displayed FDH retained its original enzymatic activity after incubation at 4°C for one month with the half-life of 17days at 40°C and 38h at 50°C. The FDH activity could be inhibited to different extents by some transition metal ions and anions. Moreover, the E. coli cells expressing FDH showed different tolerance to solvents. The recombinant whole cell exhibited high formate specificity. Finally, the E. coli cell expressing FDH was used to assay formate with a wide linear range of 5-700μM and a low limit of detection of 2μM. It is anticipated that the genetically engineered cells may have a broad application in biosensors, biofuels and cofactor regeneration system.

English

Among the enzymes of the cellulolytic complex, β-glucosidases are noteworthy due to the possibility of their application in different industrial processes, such as production of biofuels, winemaking, and development of functional foods. This study aimed to evaluate the production and characterization of β-glucosidase from the filamentous fungus Gongronella butleri, recently isolated from Cerrado soil and cultivated in agro-industrial residue substrates. The highest production of β-glucosidase, about 215.4 U/g of dry substrate (or 21.5 U/mL), was obtained by cultivation of the microorganism on wheat bran with 55% of the initial moisture, for 96 h at 30°C. This β-glucosidase showed higher catalytic activity at pH 4.5, and a temperature of 65°C. The original enzymatic activity was recovered in a pH range of 3.0-7.5 after 24 h of incubation. The enzyme retained 80% of its catalytic activity when incubated for 1 h at 50°C. The enzyme was strongly inhibited by glucose, an effect that was completely reversed by increasing substrate concentration in the reaction mixture, which is typical for competitive inhibition. High catalytic activity was observed in solutions containing up to 20% ethanol, allowing the application of this enzyme in processes with high alcohol concentrations (for example beverages and biofuels). The significant production of β-glucosidase by the selected strain, along with these enzyme characteristics, highlights the biotechnological potential of the fungus G. butleri. Key words: Microbial enzyme, biofuels, agro-industrial residues, cellulases, hemicellulases.

Response surface methodology and artificial neural network modelling of an aqueous two-phase system for purification of a recombinant alkaline active xylanase

A two-stage polyethylene glycol (PEG)-phosphate aqueous two-phase system was used for purification of a highly thermostable and alkaline active recombinant xylanase. Response surface methodology (RSM) and artificial neural network (ANN) have been used to develop predictive models for simulation and optimization of purification process. Effects of pH, PEG molecular weight, concentrations of phosphate, PEG and NaCl on the partitioning of the target enzyme and the contaminants were studied using a central composite design of experiments. The best first stage purification was achieved using 6% PEG 6000, 20% phosphate and pH 6. The optimum back extraction stage system consist of 10% phosphate, 10% NaCl, pH 10 and the first stage separation top phase. After the two stage phase separations, about 78% of the original enzyme activity was recovered and the specific activity of the purified enzyme was increased by a factor of 6.7. Also, the aqueous two-phase system was scaled-up 100 times. After back-extraction, the specific activity increased 6.56 times with 72% total yield. A similar design was also used to obtain a training set for ANN. A comparison between the model results and experimental data gave high correlation coefficient (R2) and showed that both models were able to predict the partitioning behavior. The results demonstrated a higher prediction accuracy of ANN compared to RSM. This superiority of ANN over other multi factorial approaches could make this estimation technique a very helpful tool for purification process.

Abstract A196: Stromal cells suppress cancer development by secreted GAPDH-E-cadherin interaction

Abstract The cancer tissue contains lots of stromal cells. Among them, fibroblast-like stromal cells regulate the growth of cancer cells positively and negatively through secreted factors and adhesion. We have been studying the interactions between cancer cells and stromal cells using small molecules that can modulate the interactions. We previously reported that gastric stromal cells enhance the growth of gastric cancer cells through secretion of IL-6, which is stimulated by gastric cancer cells through PGE2 and TNF-alpha reciprocally. Here we report that GAPDH, a house keeping protein, is a negative regulator of the interactions. By purifying the growth inhibitory activity against gastric cancer cells from secreted factors of gastric stromal cell we unexpectedly identified GAPDH as an active substance. While GAPDH is known to be secreted extracellularly, its growth inhibitory activity has not yet. As a result, GAPDH is found to be secreted from various organ-derived stromal cells and to inhibit the growth of various cancer cell lines. We have found that this growth inhibitory activity of GAPDH doesn't need its original enzymatic activity and GAPDH inhibits mTOR-p70S6 kinase pathway by binding to E-cadherin on cancer cell membranes. We propose that negative regulation of cancer growth using GAPDH could be a new anti-cancer strategy. Citation Format: Kawada Manabu, Junjiro Yoshida, Hiroyuki Inoue, Shun-ichi Ohba, Manabu Yamasaki, Ihomi Usami, Shuichi Sakamoto, Hikaru Abe, Takumi Watanabe, Akio Nomoto, Masakatsu Shibasaki. Stromal cells suppress cancer development by secreted GAPDH-E-cadherin interaction. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A196.

Effects of Combined High Hydrostatic Pressure and Dense Phase Carbon Dioxide on the Activity, Structure and Size of Polyphenoloxidase.

High hydrostatic pressure (HHP) may activate undesirable enzymes such as polyphenoloxidase (PPO). Carbon dioxide (CO2 ) addition to HHP could increase enzyme inactivation. We investigated the inactivation of combined HHP and dense phase carbon dioxide process on activity, secondary conformation and size of pure PPO from mushroom. Solutions (2.35μM, in phosphate buffer pH 6.8) were treated with HHP alone (HHP), or 3.6% w/w of CO2 was injected into the package (HHP+CO2). Treatment conditions were 600 MPa, 20 °C, for 1, 3, 5, 7, and 9 min. HHP+CO2 treatment significantly decreased residual enzyme activity (REA) to 30% to 12% after 1 to 9 min, respectively, whereas only HHP had no significant effect. Both HHP and HHP+CO2 treatments caused changes in secondary conformations, however HHP+CO2 changes were more extensive. Alpha-helix fractions were reduced by 32% and 41%, while β sheet, turn and unordered increased by 63% and 213%, 100% and 71%, and 118% and 82% for HHP and HHP+CO2, respectively after 9 min. The protein size in HHP+CO2 samples was 5- to 6-fold larger than that of Control and HHP treatment, and this increase was inversely correlated with REA. The best inactivation kinetics of HHP+CO2 model was the 2-fractional model with 2 simultaneous 1st-order steps, contributing 70% and 30% to original enzyme activity, with k(labile) = 12.15 min(-1) and k(stable) = 0.07 min(-1), respectively. No recovery in activity, secondary conformation and size in all samples were observed after 1-mo storage. Addition of CO2 in HHP treatment can improve enzyme inactivation, and therefore product shelf-life and quality. High hydrostatic pressure (HHP) achieves the safety of foods as a nonthermal method, but it may activate undesirable enzymes resulting in short shelf life due to, for example flavor and color changes. Our study determined that addition of CO2 to HHP has significant effects on enzyme inactivation, secondary conformational and molecular size changes of mushroom PPO. No recovery in activity, secondary conformation and size in all samples were observed after 1 mo storage at 4 °C. This combined process increases product shelf life, in addition to safety and nutrient preservation.

MUCOADHESIVE GEL WITH IMMOBILIZED LYSOZYME: PREPARATION AND PROPERTIES

The study of non-covalent immobilized lysozyme, as well as physico-chemical and biochemical properties of obtained mucoadhesive gel was the aim of the research. Lysozyme activity was determined by bacteriolytic method (Micrococcus lysodeikticus cells were a substrate). Lysozyme immobilization was conducted by the method of entrapment in gel. Enzyme/carrier interaction was studied by viscometric, spectrophotometric and spectrofluorimetric methods. Mucoadhesive gel with immobilized lysozyme, possessing antiinflammatory and antimicrobial activities, was prepared. Due to immobilization, proteinpolymer complex with the original enzymatic activity was formed. The product is characterized by high mucoadhesive properties, quantitative retaining of protein and bacteriolytic activity, prolonged release of the enzyme, improved biochemical characteristics (extended pH-activity profile, stability in acidic medium and during storage for 2 years), and it is perspective for further studies. The proposed method for lysozyme immobilization in the carboxymethyl cellulose sodium salt gel allows to obtain a stable, highly efficient product, with high adhesive properties for attachment to the mucous membranes, that is promising for use in biomedicine.

Production and properties of tragacanthin-conjugated lysozyme as a new multifunctional biopolymer

In this communication we describe preparation and characterization of an enzyme-biopolymer conjugate composed of water-soluble part of gum tragacanth and chicken egg white lysozyme (LZM) under mild Maillard reaction conditions. SDS-PAGE together with FT-IR spectroscopy revealed that Maillard reactions occurred between LZM and tragacanthin (TRG). Under optimum conditions (pH = 8.5, 60 °C, RH = 79%, 8 days), approximately 2 TRG molecules were attached to one LZM molecule. DSC analysis showed that conjugation with TRG increased denaturation temperature by 6.35 °C. The resulting conjugates were characterized using scanning electron microscopy. The modified enzyme activity retained 77% of the original enzymatic activity after 8 days. TRG-conjugated LZM exhibited improved solubility and emulsion properties as compared with the native LZM. In addition, a significant increase in foam capacity and stability of LZM-TRG conjugate was detected. Conjugate with TRG significantly improved the inhibitory effect of LZM on the growth of Staphylococcus aureus, Bacillus cereus, Escherichia coli and Salmonella typhi in a dose dependent manner such that at 4000 μg/ml, LZM-TRG conjugate inhibited S. aureus, B. cereus, E. coli and S. typhi by 90%, 80%, 50% and 40%, respectively. Taken together these results suggest that the functional properties and antimicrobial activities of LZM can be improved by conjugation with TRG. The conjugation might expand the applications of LZM as a multifunctional ingredient in food and pharmaceutical industries.

Future Applications of Apricot (Prunus Armeniaca Kaisa) ß Galactosidase in Dairy Industry

Abstract The present study demonstrates the immobilization of β galactosidase from apricots (Prunus armeniaca kaisa) on an inexpensive concanavalin A layered cellulose-alginate hybrid gel. Immobilized β galactosidase retained 78% of the initial activity after crosslinking by glutaraldehyde. It exhibited greater fraction of activity at both acidic and basic pH, and showed broad spectrum temperature optimum as compared to free enzyme. Moreover, immobilized enzyme exhibited higher thermal stability at 60°C and retained 80% of the original enzyme activity in presence of 3% galactose. The crosslinked immobilized enzyme showed improved hydrolysis of lactose from milk and whey in batch processes at 50°C as well as in continuous reactors operated at fl ow rate of 20 mL/h and 30 mL/h even after one month. Moreover, crosslinked adsorbed β galactosidase retained 76% activity even after its sixth repeated use, thereby promoting its use for lactose hydrolysis in various dairy products even for longer durations.

Purification of Lysozyme from Protein Mixtures by Solvent‐Freeze‐Out Technology

AbstractThe purification of hen egg white lysozyme from a lysozyme/crude extract mixture was carried out by the solvent‐freeze‐out (SFO) technology on the laboratory scale. Compared to previous works, this investigation focuses on the crystallization of lysozyme from a complicated system. The crystallization conditions were determined by screening tests. Based on these results, the crystallization process was performed in basic buffer solution in which the formation of tetragonal lysozyme crystals is considered to be difficult. The protein composition and purity of the crystals were evaluated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The enzymatic activity was also measured by the use of a spectrophotometer. The results show that lysozyme retained its original enzyme activity.

Catalytic behavior of lipase immobilized onto congo red and PEG-decorated particles.

Poly(ethylene glycol) (PEG)-decorated polystyrene (PS) nanoparticles with mean hydrodynamic diameter (D) and zeta–potential (ζ) of (286 ± 15) nm and (−50 ± 5) mV, respectively, were modified by the adsorption of Congo red (CR). The PS/PEG/CR particles presented D and ζ values of (290 ± 19) nm and (−36 ± 5) mV, respectively. The adsorption of lipase onto PS/PEG or PS/PEG/CR particles at (24 ± 1) °C and pH 7 changed the mean D value to (380 ± 20) and (405 ± 11) nm, respectively, and ζ value to (−32 ± 4) mV and (−25 ± 2) mV, respectively. The kinetic parameters of the hydrolysis of p-nitrophenyl butyrate were determined for free lipase, lipase immobilized onto PS/PEG and PS/PEG/CR particles. Lipase on PS/PEG/CR presented the largest Michaelis-Menten constant (KM), but also the highest Vmax and kcat values. Moreover, it could be recycled seven times, losing a maximum 10% or 30% of the original enzymatic activity at 40 °C or 25 °C, respectively. Although lipases immobilized onto PS/PEG particles presented the smallest KM values, the reactions were comparatively the slowest and recycling was not possible. Hydrolysis reactions performed in the temperature range of 25 °C to 60 °C with free lipases and lipases immobilized onto PS/PEG/CR particles presented an optimal temperature at 40 °C. At 60 °C free lipases and lipases immobilized onto PS/PEG/CR presented ~80% and ~50% of the activity measured at 40 °C, indicating good thermal stability. Bioconjugation effects between CR and lipase were evidenced by circular dichroism spectroscopy and spectrophotometry. CR molecules mediate the open state conformation of the lipase lid and favor the substrate approaching.

Dissecting Physical and Biochemical Factors of Catalytic Effectiveness in Immobilized D‐Amino Acid Oxidase by Real‐Time Sensing of O2 Availability Inside Porous Carriers

AbstractD‐Amino acid oxidase (DAAO) presents a paragon for effective use of biocatalytic O2‐dependent transformations in fine‐chemical and pharmaceutical synthesis at the industrial scale. Solid‐supported DAAO immobilizates are applied to continuous processing, but their activity and stability are often inadequate. Targeted immobilization development is restricted by insufficient knowledge of physical and biochemical factors governing the performance of heterogeneous DAAO catalysts. We have applied real‐time optical sensing of the O2 availability in luminescence‐labeled porous Sepabeads and ReliSorb carriers to quantify diffusional restrictions in DAAO immobilizates differing in the mode of enzyme attachment to the solid surface. We show that noncovalent oriented immobilization of DAAO (from Trigonopsis variabilis) resulted in high retention of the original enzyme activity (≥60 %), whereas covalent multipoint fixation caused massive (up to 90 %) activity loss. Depletion of O2 inside the solid immobilizates became limiting for enzyme catalytic effectiveness at activity loadings as low as 5 units gcarrier−1. Slow pore diffusion was principally responsible for the observed large mass‐transfer resistance, and this provides a main starting point for process intensification.

INFLUENCE OF pH AND TEMPERATURE ON THE ACTIVITY OF SnO2-BOUND α-AMYLASE: A GENOTOXICITY ASSESSMENT OF SnO2 NANOPARTICLES

Immobilization of biologically important molecules on a myriad of nanosized materials has attracted great attention due to their small size, biocompatibility, higher surface-to-volume ratio, and lower toxicity. These properties make nanoparticles (NPs) a superior matrix over bulk material for the immobilization of enzymes and proteins. In the present study, Bacillus amyloliquefaciens α-amylase was immobilized on SnO2 nanoparticles by a simple adsorption mechanism. Nanoparticle-adsorbed enzyme retained 90% of the original enzyme activity. Thermal stability of nanosupport was investigated by thermogravimetric and differential thermal analysis. Scanning electron microscopic studies showed that NPs have porous structure for the high-yield immobilization of α-amylase. The genotoxicity of SnO2-NPs was analyzed by pUC19 plasmid nicking and comet assay and revealed that no remarkable DNA damage occurred in lymphocytes. The pH-optima was found to be the same for both free and SnO2-NPs bound enzyme, while the temperature-optimum for NPs-adsorbed α-amylase was 5°C higher than its free counterpart. Immobilized enzyme retained more than 70% enzyme activity even after its eight repeated uses.